Quorum sensing is cell density-dependent regulation of genes that involves a freely diffusible molecule synthesized by the cell called an autoinducer (Fuqua et al., 1996, Salmond et al., 1995, Sitnikov et al, 1995). The paradigm system for quorum sensing is the lux system of the luminescent marine bacterium, Vibrio fischeri. V. fischeri exists at low cell densities in sea water and also at very high cell densities within the light organs of various marine organisms, such as the squid Euprymna scolopes (Pesci et al. 1997, Ruby, 1996). At high cell densities, the V. fischeri genes encoding the enzymes required for light production are expressed. These genes are part of the lux ICDABEG operon and are regulated by the gene products of luxI and luxR (Baldwin et al., 1989, Eberhard et al., 1991, Gray et al. 1992). LuxI is an autoinducer synthase that catalyzes the formation of the V. fischeri autoinducer (VAI), N-(3oxohexanoyl) homoserine lactone (Eberhard et al. 1981, Seed et al. 1995). The autoinducer freely diffuses across the cell membrane and at high cell densities, reaches a critical concentration (Kaplan et al. 1985). At this critical concentration, VAI interacts with LuxR, a DNA-binding transcriptional regulator. The LuxR-VAI complex then binds to an upstream sequence of the lux operon called the xe2x80x9clux boxxe2x80x9d, and activates transcription (Devine et al. 1989, Hanzelka et al. 1995, Stevens et al. 1994). Since one of the genes of the operon is luxI, an autoregulatory loop is formed.
Many gram-negative bacteria have been shown to possess one or more quorum sensing systems (Fuqua et al., 1996, Salmond et al., 1995). These systems regulate a variety of physiological processes, such as conjugal plasmid transfer in the plant pathogen Agrobacterium tumefaciens and antibiotic production in Erwinia stewartii. The systems typically have acylated homoserine lactone ring autoinducers, in which the homoserine lactone ring is conserved. The acyl side chain, however, can vary in length and degree of substitution. Pseudomonas aeruginosa has two quorum sensing systems, las and rhl (Brint et al. 1995, Hanzelka et al. 1996, Baldwin et al., 1989, Passador et al. 1993, Pearson et al. 1997, Pesci et al. 1997). The two systems have distinct autoinducer synthases (lasI and rhlI), transcriptional regulators (lasR and rhlR), and autoinducers (N-(3-oxododecanoyl) homoserine lactone (HSL) and N-butyryl HSL) (Sitnikov et al, 1995, Stevens et al. 1994). N-(3-oxododecanoyl) homoserine lactone is synthesized by LasI along with a small amount of N-(3-oxooctanoyl) HSL and N-(3-oxohexanoyl) HSL, while RhlI makes primarily N-butyryl HSL and a small amount of N-hexanoyl (Pearson et al. 1994, Winson et al. 1995). The rhl and las systems are involved in regulating the expression of a number of secreted virulence factors, biofilm development, and the stationary phase sigma factor (RpoS) (Brint et al. 1995, Davies et al. 1998, Latifi et al. 1996, Ochsner et al. 1995, Pesci et al. 1997). Expression of the rhl system requires a functional las system, therefore the two systems in combination with RpoS constitute a regulatory cascade (Pesci et al. 1997, Seed et al. 1995).
Quorum sensing systems are essential for communication between bacterial cells in many environments, including living biofilms. Biofilms contain distinct microcolonies, separated by discrete water channels. Biofilms are characterized by an extensive matrix of acidic polysaccharides, which protect the biofilm bacteria from biocides. In order to synthesize the polysaccharide matrix, the bacteria communicate through the quorum sensing system. Bacterial biofilms are ubiquitous and are seen in substrates ranging from sewage pipes and medical implants to teeth and the lungs of immunocompromised hosts.
The present invention is based, at least in part, on the discovery that bacterial autoinducer synthase molecules catalyze the synthesis of homoserine lactone autoinducers in a highly specific interaction of particular homoserine lactone substrates. This provided the capability to develop compositions and methods to modulate the quorum sensing capabilities of bacterial cells by controlling autoinducer production.
The present invention provides a method for identifying modulators of the autoinducer synthesis reaction, which promote or inhibit the production of homoserine lactones. Such modulators are useful for controlling bacterial growth and can be used for therapeutic treatment of bacterial infections particularly in immunocompromised subjects. They are also useful in treating disease states associated with biofilm development.
The invention pertains to methods that modulate the activity of an autoinducer synthase molecule by providing an effective amount of a compound capable of binding to the homoserine lactone substrate binding site on the autoinducer synthase molecule. The invention includes modulation of the activity of autoinducer synthase molecules including LuxI, AinS, LucM, LasI, RhlI, PhzI, TraI, HslI, EsaI, EagI, YenI, SwrI, and AhyI.
The present invention pertains to methods of selecting a compound capable of modulating the activity of the autoinducer synthase molecule by providing an effective amount of the potentially modulating compound, and determining whether the compound effectively modulates the activity of the autoinducer synthase, then selecting those compounds which do modulate the activity of the autoinducer synthase. Methods are also provided where the extent of the modulation of the activity of the autoinducer synthase is determined. A preferred embodiment pertains to a method for determining the extent of modulation by the potentially modulating compound by providing a sufficient amount of a labeled homoserine lactone substrate, allowing the reaction to go to completion, then determining the extent of the conversion of the labeled homoserine lactone substrate to homoserine lactone product.
The present invention also pertains to a method for producing highly active recombinant autoinducer synthase molecules by introducing DNA encoding the autoinducer synthase molecules into a bacterial host cell of the same species. The invention also pertains to the autoinducer synthase produced by the methods of the invention. In preferred embodiments, the purity of the autoinducer synthase is in the range from about 50-100%. In preferred embodiments, the purity of the autoinducer synthase is in the range from about 75-100%. In preferred embodiments, the purity of the autoinducer synthase is in the range from about 85-100%. In particularly preferred embodiments, the purity of the autoinducer synthase is about 95%. In a preferred embodiment, the autoinducer synthase of the RhlI quorum sensing system of P. aeruginosa is produced by the methods of the invention. In a particularly preferred embodiment, the RhlI autoinducer synthase has the amino acid sequence of SEQ ID NO 1. In a preferred embodiment, the RhlI autoinducer synthase has an active portion which includes amino acids 24-104. In a preferred embodiment, the RhlI autoinducer synthase has an active portion which includes amino acids 24-73. In a preferred embodiment, the RhlI autoinducer synthase has an amino acid sequence which includes Arginine 24, Glutamic Acid 46, Aspartic Acid 48 and Glutamic Acid 101.
The present invention also pertains to a method for modulating the formation of bacterial quorum system autoinducers by providing compounds which modulate the production of bacterial autoinducers by blocking the binding of homoserine lactone substrates to the homoserine lactone binding site on the autoinducer synthase molecules. In preferred embodiments, the autoinducer synthase molecules are RhlI.
The present invention also pertains to a method of modulating biofilm development in an immunocompromised individual by administering a therapeutically effective amount of an autoinducer synthase blocker. In preferred embodiments, the method is provided wherein the immunocompromised individual is afflicted with cystic fibrosis or HIV.
The present invention also pertains to a method of inhibiting the infectivity of a pathogenic bacteria by administering a therapeutically effective amount of an autoinducer synthase blocker molecule.
The invention pertains to a method of treating a subject for a disease state associated with biofilm development by administering a therapeutic composition of an autoinducer synthase blocker molecule and a pharmaceutically effective carrier. In a preferred embodiment, the invention pertains to a method of treating a human with cystic fibrosis or HIV.
The invention also pertains to a method of treating a subject for a state associated with autoinducer synthesis by administering an effective amount of an autoinducer synthase blocker.
The invention further pertains to a purified autoinducer synthase molecule. In preferred embodiments, the autoinducer synthase molecule is at least about 50% pure. In particularly preferred embodiments, the autoinducer synthase molecule is at least about 95% pure.
The invention also pertains to a biologically active autoinducer synthase molecule. In preferred embodiments, the autoinducer synthase molecule is substantially free of other contaminants. In particularly preferred embodiments, the autoinducer synthase molecule is substantially free of inclusion bodies.
The invention also pertains to a highly soluble autoinducer synthase molecule having biological activity.